Methicillin-resistant Staphylococcus aureus (MRSA) is a Staphylococcus aureus variant containing a Staphylococcal Chromosome Cassette (SCC) transposon that has been integrated into the genome of Staphylococcus aureus. There are at least five (5) types of MRSA; namely types I, II, III, IV and V. Types I-III are the hospital associated MRSA (HA-MRSA), with type II being the most prevalent among the HA-MRSA. Types IV-V, together with their expression of the Panton-Valentine Leukocidin (PVL) toxin, represent the community associated-MRSA (CA-MRSA). SCC in HA-MRSA is a larger mobile genetic element (when compared to CA-MRSA) that can incorporate many antibiotic resistance genes against clindamycin or doxycyline. However, CA-MRSA is more virulent than HA-MRSA. MRSA identification and SCC typing are crucial for medical diagnosis. An accurate and speedy assay to identify MRSA and its SCC types at the early stage of bacterial infection is urgently needed, so as to provide an adequate time window for proper antibiotics treatment to save human lives.
SCC integrates into the Staphylococcus aureus genome at a specific site within the orfX gene. The SCC contains numerous genes including, for example, mecA, mecR, mecI, and ccrAB. The mecA gene encodes an alternative penicillin binding protein 2 (PBP2a) that exhibits a lower affinity towards β-lactam antibiotics. The mecR gene encodes a receptor for β-lactam antibiotics, and the mecI gene encodes a transcriptional repressor for the mecA gene. Altogether, the mecA, mecR, and mecI account for the methicillin resistance for Staphylococcus aureus. The ccrAB gene is required for the transposition of the SCC mobile element.
Presently, commercial detection of MRSA involves cell culturing followed by molecular PCR technology. Specifically, Staphylococcus-containing micro-organisms are plated onto Staphylococcal selective and differential media such as Manitol Salt Agar with phenol red or Staphylococcal CHROMAgar. This cell culture procedure allows the selection of Staphylococcus aureus. 
After the culture plating, Staphylococcus aureus DNA is isolated and subjected to PCR assay for the presence of the mecA gene. mecA gene is known to exist in the SCC of MRSA, but not in methicillin-susceptible Staphylococcus aureus (MSSA). Thus, detection of the mecA gene in the chromosomal DNA of Staphylococcus aureus by PCR or hybridization makes it possible to differentiate MRSA from MSSA. Unfortunately, detection of mecA is not specific for Staphylococcus aureus, because other Staphylococcus spp. may contain SCC that contains mecA. Such Staphylococcus spp. include, for example, coagulase-negative Staphylococcus. 
Other alternative means for detecting MRSA involves PCR amplification of the 5′orfX mecDNA junction. However, mecA gene may loop out from the SCC by gene recombination. When this occurs, false positive results would ensure from the PCR amplification test. U.S. Pat. No. 6,156,507 discloses use of single-pair primers, with the forward exclusively within orf X (IntM) and the reverse within the mecA region. Because SCCmec transposon can pop out from the Staphylococcus aureus, leaving behind a portion of the right extremity (RE) region, this assay is not ideal because it often yields false-positive result. U.S. Pat. No. 2008/0220428 discloses a real-time PCR utilizing multiple primer pairs specifically targeting within orf X and mecA region and the RE region of SCCmec. This assay also yields false-positive results for the same reason.
U.S. Pat. Nos. 2007/0082340, 2008/0227087 and 2006/0252078 similarly disclose use of single-pair primers that target the SCCmec right extremity junction (MREJ) in a PCR reaction. These authors stated their tests are capable of distinguishing twenty (20) MRSA subtypes (i.e., Types I to XX). The '340 and '087 applications specifically target within SCCmec region (i.e., mecA), while '078 application targets regions extended to IntM region as well as 5′ UTR.
U.S. Pat. No. 2006/0252069 discloses a multiplex PCR assay using ccrAB primers. Because SCCmec transposon can exist in Staphylococcus aureu and non-Staphylococcus aureu bacteria (i.e., SCCmec transposon can insert into coagulase-negative Staphylococcus spp.). The '069 method cannot distinguish Staphylococcus aureus from these coagulase-negative staphylococci. A separate test (i.e., cell culture and isolation) is required for determining Staphylococcus aureus identity.
Commercial detection of MRSA involves first identification of MRSA, followed by the SCC typing. With respect to SCC typing, PCR amplification of the hypervariable regions of the ccrAB gene sequence provides useful information in differentiating types I-V. SCC typing may also be performed by PCR amplification at the left arm of the 5′orfX mecDNA junction. The major disadvantage of these procedures relates to the fact that they take a minimal time period of 24-48 hours. Because physicians are reluctant to provide any empiric therapy until the MRSA SCC typing is identified, these assays involving ccrAB gene and 5′orfX mecDNA junction are not ideal.
Current methods for distinguishing CA-MRSA from HA-MRSA involve identifying the Staphylococcal Chromosomal Cassette (SCC) mec element, together with the PVL toxin. Hence, CA-MRSA contains a SCCMec type IV cassette and possesses the PVL toxin. In contrast, HA-MRSA contains SCCMec type I-III but lacks PVL toxin. Unfortunately, MRSA PCR assays are unduly complicated. First, one must use multiple oligonucleotide primers to verify: (i) if the isolate is Staphylococcus aureus; (ii) if the isolate contains a mecA gene; and (iii) if the isolate contains a SCC Mec I-IV typing. After this PCR determination, a second PCR reaction must then be performed to show the presence or absence of PVL toxin. Because these are two separate PCR reactions, and involves the use of multiple primers and probes in respective PCR reactions, the entire procedure is both time-consuming and expensive. Such assay is overly complicated and causes delay in the diagnosis of MRSA.
A further method for determining CA-MRSA is multilocus sequence typing (MLST). In MLST, a number of housekeeping genes are sequenced and compared to reference strains. Pulsed-field gel electrophoresis (PFGE) is also used in which digested genomic DNA is separated across an agarose gel in several different orientations to gain resolution of large bands of DNA. Both MLST and PFGE are time-consuming and require a high degree of skill in order to successfully interpret results, making them less than optimal for use in medical diagnostics.
All these prior art methodologies suffer either suboptimal specificity or unacceptable long assay time. Accordingly, there is a continuing need for an accurate, rapid and simple PCR assay to detect MRSA and simultaneously determine its SCC types (i.e., types I, II, III, IV or V), preferably in a single PCR reaction.